Requirement for PINCH in Skeletal Myoblast Differentiation

Abstract Background: Skeletal muscle is composed of bundles of myofibers ensheathed by extracellular matrix networks. Malformation of skeletal muscle during embryonic development results in congenital myopathies. Disease mechanisms of congenital myopathies remain unclear. PINCH, an adaptor of focal adhesion complex, plays essential roles in multiple cellular processes and organogenesis. Elucidation of the molecular mechanisms underlying skeletal myogenesis will offer new insights into pathogenesis of myopathies.Methods: We generated muscle-specific PINCH knock-out mice to study the functional role of PINCH in skeletal myogenesis. Histologic and Transmission Electron Microscopy analysis demonstrated that Impaired myogenic differentiation and maturation in mice with PINCH1 being ablated in skeletal muscle progenitors, and Ablation of PINCH1 and PINCH2 resulted in reduced size of muscle fibers and impaired multinucleation; Cell culture and immunostaining showed that defects in myoblast fusion and cytoskeleton assembly in PINCH double mutant mice; Western blotting showed that defects in expression of cytoskeleton proteins and proteins involved in myogenesis in DMUT skeletal muscles.Results: Double ablation of PINCH1 and PINCH2 resulted in early postnatal lethality with reduced size of skeletal muscles and detachment of diaphragm muscles from the body wall. Myofibers of PINCH mutant myofibers failed to undergo multinucleation and exhibited disrupted sarcomere structures. The mutant myoblasts in culture were able to adhere to newly formed myotubes, but impeded in cell fusion and subsequent sarcomere genesis and cytoskeleton organization. Consistent with this, expression of integrin β1 and some cytoskeleton proteins, and phosphorylation of ERK and AKT were significantly reduced in PINCH mutants. Expression of MRF4, the most highly expressed myogenic factor at late stages of myogenesis, was abolished in PINCH mutants, that could contribute to observed phenotypes. In addition, mice with PINCH1 being ablated in myogenic progenitors exhibited only mild centronuclear myopathic changes, suggesting a compensatory role of PINCH2 in myogenic differentiation, indicating a critical role of PINCH proteins in myogenic differentiation.Conclusion: Our results demonstrated an essential role of PINCH in skeletal myogenic differentiation.